A lack of geographic and racial diversity in clinical trial populations may arise from a disproportionate focus on trial conduct in the US and Europe. We sought to assess the proportion of leaders and collaborators from seminal cardiometabolic trials from the Asia-Pacific (APAC) region.

The inwardly rectifying potassium current of the cardiomyocyte (I K1 ) is the main determinant of the resting potential. Ion channels Kir2.1, Kir2.2, and Kir2.3 form tetramers and are the molecular correlate of macroscopic I K1 current. Verapamil is an antiarrhythmic drug used to suppress atrial and ventricular arrhythmias. Its primary mechanism of action is via blocking calcium channels. In addition, it has been demonstrated to block I K1 current and the Kir2.1 subunit. Its effect on other subunits that contribute to I K1 current has not been studied to date. We therefore analyzed the effect of verapamil on the Kir channels 2.1, 2.2, and 2.3 in the Xenopus oocyte expression system. Kir2.1, Kir2.2, and Kir2.3 channels were heterologously expressed in Xenopus oocytes. Respective currents were measured with the voltage clamp technique and the effect of verapamil on the current was measured. At a concentration of 300 µM, verapamil inhibited Kir2.1 channels by 41.36% ± 2.7 of the initial current, Kir2.2 channels by 16.51 ± 3.6%, and Kir2.3 by 69.98 ± 4.2%. As a verapamil effect on kir2.3 was a previously unknown finding, we analyzed this effect further. At wash in with 300 µM verapamil, the maximal effect was seen within 20 min of the infusion. After washing out with control solution, there was only a partial current recovery. The current reduction from verapamil was the same at − 120 mV (73.2 ± 3.7%), − 40 mV (85.5 ± 6.5%), and 0 mV (61.5 ± 10.6%) implying no voltage dependency of the block. Using site directed mutations in putative binding sites, we demonstrated a decrease of effect with pore mutant E291A and absence of verapamil effect for D251A. With mutant I214L, which shows a stronger affinity for PIP 2 binding, we observed a normalized current reduction to 61.9 ± 0.06% of the control current, which was significantly less pronounced compared to wild type channels. Verapamil blocks Kir2.1, Kir2.2, and Kir2.3 subunits. In Kir2.3, blockade is dependent on sites E291 and D251 and interferes with activation of the channel via PIP 2 . Interference with these sites and with PIP 2 binding has also been described for other Kir channels blocking drugs. As Kir2.3 is preferentially expressed in atrium, a selective Kir2.3 blocking agent would constitute an interesting antiarrhythmic concept.

Postcardiac injury syndrome (PCIS) is an inflammatory complication that derives from injury to the epicardium, myocardium, or endocardium. It occurs after trauma, myocardial infarction, percutaneous coronary intervention, cardiac surgery, intracardiac ablation, and implantation of cardiac implantable electronic device (CIED). In this study we assessed the incidence of PCIS after CIED implantation and its possible risk factors. All patients who received CIED implantation at Heidelberg University Hospital between 2000 and 2014 were evaluated (n = 4989 patients). Clinical data including age, sex, underlying cardiac disease, type of implanted CIED, location of electrode implantation, clinical symptoms, time of symptom onset of PCIS, therapy, and outcome were extracted and analyzed. We identified 19 cases of PCIS in 4989 patients, yielding an incidence of 0.38%. The age of patients with PCIS ranged from 39 to 86 years. Dilated cardiomyopathy (DCM) as underlying cardiac disease and right atrial (RA) lead implantation had a significant association with occurrence of PCIS (p = 0.045 in DCM and p < 0.001 in RA lead implantation). Dyspnea, chest pain, dry cough, and fever were the most frequently reported symptoms in patients with PCIS. Pericardial and pleura effusion as well as elevated C‑reactive protein (CRP), increased erythrocyte sedimentation rate (ESR), and leukocytosis were the most common findings. To the best of our knowledge, this is the largest cohort evaluating the incidence of PCIS after CIED implantation. The data show that PCIS is a rare complication after CIED implantation and occurs more frequently in patients with DCM and those with RA lead implantation. Although rare and mostly benign, PCIS can lead to potentially lethal complications and physicians must be aware of its symptoms.

Left bundle branch block (LBBB) has a predictive value for response to cardiac resynchronization therapy as reported by Zareba et al. (Circulation 123(10):1061–1072, 2011). However, based on ECG criteria, the discrimination between complete LBBB and nonspecific intraventricular conduction delay is challenging. We tested the hypothesis that discrimination can be performed using standard electrophysiological catheters and a simple stimulation protocol.

There is genetic and epidemiological evidence demonstrating the association between Lp(a) levels and cardiovascular disease. While evolocumab reduces Lp(a) by 25-30%, the impact of PCSK9 inhibition on plaque at different Lp(a) levels is unknown. GLAGOV compared the effects of the PCSK9 inhibitor,

The cardiac IK1 current stabilizes the resting membrane potential of cardiomyocytes. Protein kinase A (PKA) induces an inhibition of IK1 current which strongly promotes focal arrhythmogenesis. The molecular mechanisms underlying this regulation have only partially been elucidated yet. Furthermore, the role of A-kinase anchoring proteins (AKAPs) in this regulation has not been examined to date. The objective of this project was to elucidate the molecular mechanisms underlying the inhibition of IK1 by PKA and to identify novel molecular targets for antiarrhythmic therapy downstream β-adrenoreceptors. Patch clamp and voltage clamp experiments were used to record currents and co-immunoprecipitation, and co-localization experiments were performed to show spatial and functional coupling. Activation of PKA inhibited IK1 current in rat cardiomyocytes. This regulation was markedly attenuated by disrupting PKA-binding to AKAPs with the peptide inhibitor AKAP-IS. We observed functional and spatial coupling of the plasma membrane-associated AKAP15 and AKAP79 to Kir2.1 and Kir2.2 channel subunits, but not to Kir2.3 channels. In contrast, AKAPyotiao had no functional effect on the PKA regulation of Kir channels. AKAP15 and AKAP79 co-immunoprecipitated with and co-localized to Kir2.1 and Kir2.2 channel subunits in ventricular cardiomyocytes. In this study, we provide evidence for coupling of cardiac Kir2.1 and Kir2.2 subunits with the plasma membrane-bound AKAPs 15 and 79. Cardiac membrane-associated AKAPs are a functionally essential part of the regulatory cascade determining IK1 current function and may be novel molecular targets for antiarrhythmic therapy downstream from β-adrenoreceptors.

Class IA antiarrhythmic drug quinidine was one of the first clinically used compounds to terminate atrial fibrillation and acts as multichannel inhibitor with well-documented inhibitory effects on several cardiac potassium channels. In the mammalian heart, heteromeric assembly of Kir2.1-2.3 channels underlies IK1 current. Although a low-affinity block of quinidine on Kir2.1 has already been described, a comparative analysis of effects on other Kir2.x channels has not been performed to date. Therefore, we analyzed the effects of quinidine on wild-type and mutant Kir2.x channels in the Xenopus oocyte expression system. Quinidine exerted differential inhibitory effects on Kir2.x channels with the highest affinity toward Kir2.3 subunits. Onset of block was slow and solely reversible in Kir2.2 subunits. Quinidine inhibited Kir2.x currents in a voltage-independent manner. By means of comparative Ala-scanning mutagenesis, we further found that residues E224, F254, D259, and E299 are essential for quinidine block in Kir2.1 subunits. Analogously, quinidine mediated Kir2.3 inhibition by binding corresponding residues E216, D247, D251, and E291. In contrast, Kir2.2 current block merely involved corresponding residue D260. Using channel mutants with altered (phosphatidylinositol 4,5-bisphosphate PIP2) affinities, we were able to demonstrate that high PIP2 affinities (i.e., Kir2.3 I214L) correlate with low quinidine sensitivity. Inversely, mutant channels interacting only weakly with PIP2 (i.e., Kir2.1 K182Q, and L221I) are prone to a higher inhibitory effect. Thus, we conclude that inhibition of Kir2.x channels by quinidine is mediated by joint modes of action involving direct cytoplasmic pore block and an impaired channel stabilization via interference with PIP2.

The caged xanthone gambogic acid (GA) is a novel anti-cancer agent which exhibits anti-proliferative, anti-inflammatory and cytotoxic effects in many types of cancer tissues. In a recent phase IIa study, GA exhibits a favourable safety profile. However, limited data are available concerning its interaction with cardiac ion channels. Heteromeric assembly of Kir2.x channels underlies the cardiac inwardly rectifying IK1 current which is responsible for the stabilization of the diastolic resting membrane potential. Inhibition of the cardiac IK1 current may lead to ventricular arrhythmia due to delayed afterdepolarizations. Compared to Kv2.1, hERG and Kir1.1, a slow, delayed inhibition of Kir2.1 channels by GA in a mammalian cell line was reported before but no data exist in literature concerning action of GA on homomeric Kir2.2 and Kir2.3 and heteromeric Kir2.x channels. Therefore, the aim of this study was to provide comparative data on the effect of GA on homomeric and heteromeric Kir2.x channels. Homomeric and heteromeric Kir2.x channels were heterologously expressed in Xenopus oocytes, and the two-microelectrode voltage-clamp technique was used to record Kir2.x currents. To investigate the mechanism of the channel inhibition by GA, alanine-mutated Kir2.x channels with modifications in the channels pore region or at phosphatidylinositol 4,5-bisphosphate (PIP2)-binding sites were employed. GA caused a slow inhibition of homomeric and heteromeric Kir2.x channels at low micromolar concentrations (with IC50 Kir2.1/2.2 < Kir2.2 < Kir2.2/2.3 < Kir2.3 < Kir2.1 < Kir2.1/2.3). The effect did not reach saturation within 60 min and was not reversible upon washout for 30 min. The inhibition showed no strong voltage dependence. We provide evidence for a combination of direct channel pore blockade and a PIP2-dependent mechanism as a molecular basis for the observed effect. We conclude that Kir2.x channel inhibition by GA may be relevant in patients with pre-existing cardiac disorders such as chronic heart failure or certain rhythm disorders and recommend a close cardiac monitoring for those patients when treated with GA.

BACKGROUND:Cardiac inwardly rectifying Kir current (IK1) mediates terminal repolarisation and is critical for the stabilization of the diastolic membrane potential. Its predominant molecular basis in mammalian ventricle is heterotetrameric assembly of Kir2.1 and Kir2.2 channel subunits. It has been shown that PKC inhibition of IK1 promotes focal ventricular ectopy. However, the underlying molecular mechanism has not been fully elucidated to date.METHODS AND RESULTS:In the Xenopus oocyte expression system, we observed a pronounced PKC-induced inhibition of Kir2.2 but not Kir2.1 currents. The PKC regulation of Kir2.2 could be reproduced by an activator of conventional PKC isoforms and antagonized by pharmacological inhibition of PKCβ. In isolated ventricular cardiomyocytes (rat, mouse), pharmacological activation of conventional PKC isoforms induced a pronounced inhibition of IK1. The PKC effect in rat ventricular cardiomyocytes was markedly attenuated following co-application of a small molecule inhibitor of PKCβ. Underlining the critical role of PKCβ, the PKC-induced inhibition of IK1 was absent in homozygous PKCβ knockout-mice. After heterologous expression of Kir2.1-Kir2.2 concatemers in Xenopus oocytes, heteromeric Kir2.1/Kir2.2 currents were also inhibited following activation of PKC.CONCLUSION:We conclude that inhibition of cardiac IK1 by PKC critically depends on the PKCβ isoform and Kir2.2 subunits. This regulation represents a potential novel target for the antiarrhythmic therapy of focal ventricular arrhythmias.

Midazolam is a short-acting benzodiazepine that is in wide clinical use as an anxiolytic, sedative, hypnotic, and anticonvulsant. Midazolam has been shown to inhibit ion channels, including calcium and potassium channels. So far, the effects of midazolam on cardiac human ether-à-go-go-related gene (hERG) channels have not been analyzed. The inhibitory effects of midazolam on heterologously expressed hERG channels were analyzed in Xenopus oocytes using the double-electrode voltage clamp technique. We found that midazolam inhibits hERG channels in a concentration-dependent manner, yielding an IC50 of 170 μM in Xenopus oocytes. When analyzed in a HEK 293 cell line using the patch-clamp technique, the IC50 was 13.6 μM. Midazolam resulted in a small negative shift of the activation curve of hERG channels. However, steady-state inactivation was not significantly affected. We further show that inhibition is state-dependent, occurring within the open and inactivated but not in the closed state. There was no frequency dependence of block. Using the hERG pore mutants F656A and Y652A we provide evidence that midazolam uses a classical binding site within the channel pore. Analyzing the subacute effects of midazolam on hERG channel trafficking, we further found that midazolam does not affect channel surface expression. Taken together, we show that the anesthetic midazolam is a low-affinity inhibitor of cardiac hERG channels without additional effects on channel surface expression. These data add to the current understanding of the pharmacological profile of the anesthetic midazolam.

Midazolam is a short-acting benzodiazepine that is in wide clinical use as an anxiolytic, sedative, hypnotic, and anticonvulsant. Midazolam has been shown to inhibit ion channels, including calcium and potassium channels. So far, the effects of midazolam on cardiac human ether-à-go-go-related gene (hERG) channels have not been analyzed. The inhibitory effects of midazolam on heterologously expressed hERG channels were analyzed in Xenopus oocytes using the double-electrode voltage clamp technique. We found that midazolam inhibits hERG channels in a concentration-dependent manner, yielding an IC50 of 170 μM in Xenopus oocytes. When analyzed in a HEK 293 cell line using the patch-clamp technique, the IC50 was 13.6 μM. Midazolam resulted in a small negative shift of the activation curve of hERG channels. However, steady-state inactivation was not significantly affected. We further show that inhibition is state-dependent, occurring within the open and inactivated but not in the closed state. There was no frequency dependence of block. Using the hERG pore mutants F656A and Y652A we provide evidence that midazolam uses a classical binding site within the channel pore. Analyzing the subacute effects of midazolam on hERG channel trafficking, we further found that midazolam does not affect channel surface expression. Taken together, we show that the anesthetic midazolam is a low-affinity inhibitor of cardiac hERG channels without additional effects on channel surface expression. These data add to the current understanding of the pharmacological profile of the anesthetic midazolam.

Dronedarone is a novel class III antiarrhythmic drug that is widely used in atrial fibrillation. It has been shown in native cardiomyocytes that dronedarone inhibits cardiac inwardly rectifying current I-K1 at high concentrations, which may contribute both its antifibrillatory efficacy and its potential proarrhythmic side effects. However, the underlying mechanism has not been studied in further detail to date. In the mammalian heart, heterotetrameric assembly of Kir2.x channels is the molecular basis of I-K1 current. Therefore, we studied the effects of dronedarone on wild-type and mutant Kir2.x channels in the Xenopus oocyte expression system. Dronedarone inhibited Kir2.1 currents but had no effect on Kir2.2 or Kir2.3 currents. Onset of block was slow but completely reversible upon washout. Blockade of Kir2.1 channels did not exhibit strong voltage dependence or frequency dependence. In a screening with different Kir2.1 mutants lacking specific binding sites within the cytoplasmic pore region, we found that residue E224 is essential for binding of dronedarone to Kir2.1 channels. In conclusion, direct block of Kir2.1 channel subunits by dronedarone through binding at E224 may underlie its inhibitory effects on cardiac I-K1 current.

Midazolam is a short-acting benzodiazepine that is widely used in anesthesia. Despite its widespread clinical use, detailed information about cardiac side effects of midazolam is largely lacking. Using the double-electrode voltage clamp technique, we studied pharmacological effects of midazolam on heterologously expressed Kv1.5 channels underlying atrial repolarizing current I(Kur). Midazolam dose-dependently inhibited Kv1.5 current, yielding an IC50 of 17 μM in an HEK cell line and an IC50 of 104 μM in Xenopus oocytes. We further showed that midazolam did not affect the half-maximal activation voltage of Kv1.5 channels. However, a small negative shift of the inactivation curve could be observed. Midazolam acted as a typical open-channel inhibitor with rapid onset of block and without frequency dependence of block. Taken together, midazolam is an open channel inhibitor of cardiac Kv1.5 channels. These data add to the current understanding of the pharmacological profile of midazolam.

Objectives This study sought to test the hypothesis that inducing an autoimmune response against the cardiac sodium channel (Na(V)1.5) induces arrhythmias.Background Sporadic evidence supports the concept that autoantibodies may cause cardiac arrhythmias but substantial experimental investigations using in vivo models have been lacking to date. The Na(V)1.5 is essential for cardiac impulse propagation and its dysfunction has been linked to conduction disease.Methods Rats were immunized with a peptide sequence derived from the third extracellular loop of the first domain of Na(V)1.5. After 28 days, we evaluated in vivo both the electrical and mechanical parameters of cardiac function. Histopathology, myocardial gene and protein expression were assessed. Whole-cell patch-clamp was used to measure sodium current (I-Na) density in isolated cardiomyocytes.Results Na(V)1.5-immunized rats had high titers of autoantibodies against Na(V)1.5. On ECG recording, Na(V)1.5-immunized animals showed significantly prolonged PR-intervals. During Holter ECG-monitoring we observed repeated prolonged episodes of third-degree atrioventricular and sinoatrial block in every Na(V)1.5-immunized animal, but not in controls. Immunization had no effect on cardiac function. In comparison to controls, myocardial Na(V)1.5 mRNA and protein levels were decreased in immunized rats. I-Na density was reduced in cardiomyocytes incubated with sera from Na(V)1.5-immunized rats and from patients with idiopathic atrioventricular block (AVB) in comparison to sera from respective controls. In patients with idiopathic AVB, we observed autoantibodies against Na(V)1.5 that were absent in sera from healthy controls.Conclusions Provocation of an autoimmune response against Na(V)1.5 induces conductance defects probably caused by a reduced expression level and an inhibition of Na(V)1.5 by autoantibodies, resulting in decreased I-Na. (C) 2013 by the American College of Cardiology Foundation

Kir2.x channels form the molecular basis of cardiac I-K1 current and play a major role in cardiac electrophysiology. However, there is a substantial lack of selective Kir2 antagonists. We found the beta(3)-adrenoceptor antagonist SR59230A to be an inhibitor of Kir2.x channels. Therefore, we characterized the effects of SR59230A on Kir2.x and other relevant cardiac potassium channels.Cloned channels were expressed in the Xenopus oocyte expression system and measured with the double-microelectrode voltage clamp technique.SR59230A inhibited homomeric Kir2.1 channels with an IC50 of 33 mu M. Homomeric Kir2.2 and Kir2.3 channels and Kir2.x heteromers were also inhibited by SR59230A with similar potency. In contrast, no relevant inhibitory effects of SR59230A were found in cardiac Kv1.5, Kv4.3 and KyLQT1/minK channels. In hERG channels, SR59230A only induced a weak inhibition at a high concentration.These findings establish SR59230A as a novel inhibitor of Kir2.1-2.3 channels with a favorable profile with respect to additional effects on other cardiac repolarizing potassium channels. (C) 2012 Elsevier Inc. All rights reserved.

The zebrafish is increasingly recognized as an animal model for the analysis of hERG-related diseases. However, functional properties of the zebrafish orthologue of hERG have not been analyzed yet. We heterologously expressed cloned ERG channels in Xenopus oocytes and analyzed biophysical properties using the voltage clamp technique. zERG channels conduct rapidly activating and inactivating potassium currents. However, compared to hERG, the half-maximal activation voltage of zERG current is shifted towards more positive potentials and the half maximal steady-state inactivation voltage is shifted towards more negative potentials. zERG channel activation is delayed and channel deactivation is accelerated significantly. However, time course of zERG conducted current under action potential clamp is highly similar to the human orthologue. In summary, we show that ERG channels in zebrafish exhibit biophysical properties similar to the human orthologue. Considering the conserved channel function, the zebrafish represents a valuable model to investigate human ERG channel related diseases.

Background: Benefit from Implantable Cardiac Defibrillators (ICDs) is governed by two mutually exclusive events: a patient either benefits from potentially life saving device therapy, or is lost to death prior to initial device therapy. Current guidelines remain imperfect with regards to optimising patient selection and avoidance of unnecessary implantation-related morbidity and cost.

Background and purpose: Atomoxetine is a selective noradrenaline reuptake inhibitor, recently approved for the treatment of attention‐deficit/hyperactivity disorder. So far, atomoxetine has been shown to be well tolerated, and cardiovascular effects were found to be negligible. However, two independent cases of QT interval prolongation, associated with atomoxetine overdose, have been reported recently. We therefore analysed acute and subacute effects of atomoxetine on cloned human Ether‐à‐Go‐Go ‐Related Gene (hERG) channels. Experimental approach: hERG channels were heterologously expressed in Xenopus oocytes and in a human embryonic kidney cell line and hERG currents were measured using voltage clamp and patch clamp techniques. Action potential recordings were made in isolated guinea‐pig cardiomyocytes. Gene expression and channel surface expression were analysed using quantitative reverse transcriptase polymerase chain reaction, Western blot and the patch clamp techniques. Key results: In human embryonic kidney cells, atomoxetine inhibited hERG current with an IC 50 of 6.3 µmol·L −1 . Development of block and washout were fast. Channel activation and inactivation were not affected. Inhibition was state‐dependent, suggesting an open channel block. No use‐dependence was observed. Inhibitory effects of atomoxetine were attenuated in the pore mutants Y652A and F656A. In guinea‐pig cardiomyocytes, atomoxetine lengthened action potential duration without inducing action potential triangulation. Overnight incubation with high atomoxetine concentrations resulted in a decrease of channel surface expression. Conclusions and implications: Whereas subacute effects of atomoxetine seem negligible under therapeutically relevant concentrations, hERG channel block should be considered in cases of atomoxetine overdose and when administering atomoxetine to patients at increased risk for the development of acquired long‐QT syndrome.

The antidepressant mianserin exhibits a tetracyclic structure that is different from typical tricyclic antidepressants (TCA) and that of selective serotonin reuptake inhibitors. In comparison to the older TCA, mianserin has been shown to have a superior risk profile regarding proarrhythmic effects, both in vitro and in vivo. However, the underlying molecular electrophysiological basis has not been elucidated to date. Therefore, we studied the effects of mianserin on cardiac hERG potassium channels, the predominant target of drug-induced proarrhythmia. HERG channels were expressed in the Xenopus oocyte expression system and in human embryonic kidney (HEK) cells and currents were measured with two-microelectrode voltage-clamp and whole-cell patch-clamp, respectively. Mianserin inhibited hERG currents in a dose-dependent manner with an IC50 of 3.2 μmol/l in HEK cells. Onset of blockade was slow and the inhibitory effect was not reversible upon wash-out of the drug. In hERG channel mutants, Y652A and F656A, lacking aromatic residues in the S6 domain, the effect of mianserin was significantly reduced in comparison to the wild type. Mianserin inhibited hERG currents in the open and inactivated state, but not in the closed states. HERG inactivation kinetics were significantly altered by mianserin without marked effects on channel activation kinetics. The inhibitory effect was not frequency dependent. In conclusion, mianserin is a low-affinity hERG-blocking agent. However, taken together with the lack of APD-prolongation shown in other studies, mianserin seems to have a good safety profile. Lack of consistent QT prolonging effects of mianserin in previous studies may therefore be linked to additional effects such as inhibition of other cardiac ion channels. However, as demonstrated by clinical case reports, mianserin can induce proarrhythmic effects in susceptible patients. Therefore, in patients with complex co-medication (i.e., additional hERG-blocking agents) and in patients with risk factors for acquired long QT syndrome as well as in cases of overdose, adequate monitoring should be recommended.